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ADCY9 / Adenylate cyclase type 9 · IHC design guide
Design Immunohistochemistry for ADCY9
Plan ADCY9 chromogenic IHC in paraffin sections using the observed cytoplasmic tissue pattern alongside its annotated membrane location (HPA tissue IHC; UniProt). The catalog antibody has an IHC dilution range of 1:50–1:200; compare staining with matched controls and score the relevant cell population (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ADCY9 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Mostly cytoplasmic in tissue (HPA tissue IHC); membrane protein (UniProt) | |
| Staining pattern | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Gallbladder+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Smooth muscle may be unstained despite reported airway expression (HPA tissue IHC; UniProt) | |
| Regulation | Regulation of abundance is unreported (UniProt) | |
| Isoform / epitope | No isoforms annotated; epitope side of membrane is unspecified (UniProt) |
Section 1
Recommended ADCY9 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published ADCY9 staining protocols for colon cancer tissue arrays (PMC5759510) and lung adenocarcinoma sections (PMC10089850).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A06038-1) |
| Fixation | Image fixative and duration unreported (datasheet A06038-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-ADCY9, 1:50-1:200 (datasheet A06038-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ADCY9-positive staining in glandular cells of gallbladder (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min for membrane ADCY9 (UniProt O60503 topology; page retrieval rule).
Section 2
What Is the Expected ADCY9 Staining Pattern?
ADCY9 is a cell-membrane protein with 12 transmembrane segments (UniProt O60503 topology). In paraffin-section IHC, expect cellular staining in gallbladder glandular cells, with moderate staining in several other listed cell types (HPA tissue IHC: High in gallbladder glandular cells; Medium in adrenal, appendix, breast, colon and duodenal glandular cells, bone-marrow hematopoietic cells and cerebral-cortex glial cells). HPA describes the tissue pattern as cytoplasmic; its IHC reliability is Approved, with medium RNA concordance and external verification pending (HPA tissue IHC).
| Membranous or cytoplasmic chromogenic staining in gallbladder glandular cells, stronger than nearby unstained cells. | This fits a useful positive control: HPA scores these cells High and describes cytoplasmic expression in most tissues (HPA tissue IHC). A membrane component is consistent with ADCY9 topology, but routine IHC may not resolve a sharp membrane rim (UniProt O60503 topology; general IHC practice). |
| Signal confined to nuclei, with no convincing membrane or cytoplasmic staining. | An exclusively nuclear pattern is unsupported by UniProt cell-membrane annotation and HPA's tissue and ICC-IF localisation summaries (UniProt O60503 subcellular location; HPA tissue IHC; HPA subcellular). Treat it as suspect and compare with a positive tissue and detection controls (general IHC practice). |
| Strong staining in adipocytes, prostate glandular cells or heart cardiomyocytes. | HPA reports these specific cells as Not detected, so strong signal calls for a specificity or endogenous-detection check (HPA tissue IHC; general IHC practice). UniProt detection in heart is tissue-level evidence; it does not establish staining in cardiomyocytes (UniProt O60503 tissue specificity; HPA tissue IHC). |
| Uniform haze across cells and surrounding tissue, with little cell-specific contrast. | This is difficult to score as ADCY9 because the HPA reference identifies particular positive and negative cell populations (HPA tissue IHC). Check background in a no-primary control, then review blocking, washes and detection chemistry (general IHC practice). |
| No visible signal in gallbladder glandular cells. | HPA rates these cells High, so an entirely blank positive control calls the run into question (HPA tissue IHC). Check tissue preservation, retrieval, antibody dilution and detection with appropriate controls before interpreting other tissues as ADCY9-negative (general IHC practice). |
💡Expected ADCY9 appearanceA convincing IHC-positive section shows cell-associated membrane-compatible and/or cytoplasmic chromogen in gallbladder glandular cells, potentially High relative to HPA-listed negative cells; isolated nuclear staining or uniform background is suspect (UniProt O60503 topology; HPA tissue IHC; general IHC practice).
| Compartment and epitope access | ADCY9 has 12 membrane spans and large cytoplasmic regions, but the supplied record gives no antibody epitope (UniProt O60503 topology; HPA antibodies). Do not infer which side of the membrane the IHC-validated antibody recognizes or prescribe an ADCY9-specific retrieval condition. |
| Cell-specific tissue comparisons | HPA scores gallbladder glandular cells High, several other listed cell populations Medium, and adipocytes and prostate glandular cells Not detected (HPA tissue IHC). Use the named cells as comparators; a whole-tissue label can conceal differences among its cell types (general IHC practice). |
| Strength of validation | The two listed antibodies are IHC Approved; the tissue profile has medium staining–RNA consistency and awaits external verification (HPA antibodies; HPA tissue IHC). Interpret a matched pattern as supportive rather than definitive proof of antigen identity. |
| IF/ICC: what pattern should be expected? | HPA supports plasma-membrane localisation, marks cytosol uncertain, and lists primary cilium and basal body as uncertain additional locations (HPA subcellular). That ICC-IF evidence can guide compartment interpretation; it does not supply an IF protocol for this IHC section. |
| Retrieval and detection | No ADCY9-specific retrieval or fixation effect is established by these sources (UniProt O60503; HPA tissue IHC). When signal or background is problematic, optimize retrieval and inspect no-primary controls as general paraffin-IHC practice, without attributing an outcome to ADCY9-specific fixation sensitivity. |
| Situation | Likely cause | Next action |
|---|---|---|
| Gallbladder positive control is blank. | The run may have inadequate antigen exposure or detection; HPA expects High staining in gallbladder glandular cells (HPA tissue IHC; general IHC practice). | Verify tissue and control performance, then review the catalog antibody's IHC-P conditions, retrieval and detection steps (general IHC practice). Do not assign an ADCY9-specific fixation effect from these sources. |
| Only a dark nuclear signal is present. | An isolated nuclear compartment conflicts with UniProt cell-membrane annotation and HPA localisation summaries (UniProt O60503 subcellular location; HPA tissue IHC; HPA subcellular). | Inspect a no-primary control and a HPA-listed positive cell population; reassess specificity before scoring nuclei as positive (general IHC practice). |
| Adipocytes or prostate glandular cells stain strongly. | HPA lists those cell populations as Not detected, raising concern about nonspecific binding or endogenous detection activity (HPA tissue IHC; general IHC practice). | Compare a no-primary control, review blocking and detection chemistry, and score the named cells separately from neighboring structures (general IHC practice). |
| Cardiomyocytes stain despite a weak gallbladder control. | HPA lists cardiomyocytes as Not detected; UniProt's detection in heart does not identify the positive heart cell type (HPA tissue IHC; UniProt O60503 tissue specificity). | Resolve the failed positive control first, then compare cardiomyocyte signal with no-primary and adjacent-cell controls (general IHC practice). |
| The whole section has diffuse brown background. | Broad haze obscures the cell-specific pattern described by HPA and can arise from staining or detection background (HPA tissue IHC; general IHC practice). | Check no-primary signal, endogenous activity controls where relevant, blocking, washes and chromogen development before assigning cellular scores (general IHC practice). |
| Cytoplasmic IHC appears broader than membrane staining seen by IF/ICC. | HPA describes cytoplasmic expression in tissue IHC, while its ICC-IF plasma-membrane location is supported and cytosol is uncertain (HPA tissue IHC; HPA subcellular). | Compare cell-specific patterns within each assay and check IHC background controls; do not require identical compartment resolution across chromogenic IHC and ICC-IF (general IHC practice). |
Experiment evidence
IHC & IF images showing ADCY9 staining
A06038-1 · IHCImmunohistochemistry (IHC) analyzes of ADCY9 pAb in paraffin-embedded human breast carcinoma tissue at 1:50,showing cytoplasm and membrane s
A06038-1 · IHCImmunohistochemistry (IHC) analyzes of ADCY9 pAb in paraffin-embedded human stomach carcinoma tissue at 1:50,showing cytoplasm and membrane
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000162104-ADCY9/tissue/cerebral+cortex#imid_10697743
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000162104-ADCY9/tissue/colon#imid_10697669
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000162104-ADCY9/tissue/liver#imid_10697678
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000162104-ADCY9/tissue/kidney#imid_10697687
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA041328; green = ADCY9, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA041328; green = ADCY9, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ADCY9 IHC & IF
🧪Run gallbladder first; its glandular cells should stain (HPA: High). Run adipose tissue as the negative comparator, focusing on adipocytes (HPA: Not detected); on the gallbladder slide, treat neighboring non-glandular cells as internal background only if they lack specific staining, since their ADCY9 status is not established by the supplied HPA row.
Positive control tissue: Gallbladder (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ADCY9 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), KOLF2.1J, with annotated localisation: Plasma membrane (supported), Cytosol (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and matched rabbit IgG isotype control (caption: ADCY9 pAb with goat anti-rabbit secondary), plus ADCY9 knockout material or peptide competition if a validated blocking peptide is available. Quench endogenous peroxidase and check for endogenous biotin on gallbladder sections when using avidin–peroxidase detection (caption: avidin–peroxidase).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A06038-1 paraffin-section caption does not state the fixative (caption: fixative unreported). Retrieval dependence is unreported, so optimize antigen retrieval empirically; HPA ICC-IF images support IF assessment but do not establish that IF or frozen sections are easier than paraffin IHC (HPA: ICC-IF images; caption: paraffin section). Bile pigment may complicate chromogen scoring in gallbladder; compare with the no-primary control.
HPA tissue IHC evidence for ADCY9
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Prostate | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ADCY9 IHC Tips
Troubleshoot ADCY9 staining in paraffin sections by checking retrieval, compartment, cell type, and controls before comparing chromogenic scores (UniProt O60503; HPA tissue IHC).
How should I retrieve ADCY9 in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0, 95–98 °C for 20 min (page retrieval setting). Cool sections in buffer before washing, and compare a dilution series against the catalog antibody’s 1:50 paraffin-section example (selected-SKU caption; standard IHC practice). If staining remains weak, trial a different retrieval buffer on adjacent sections while holding antibody concentration and detection constant (standard IHC practice). Record whether glandular-cell staining improves without a matching rise in background, using the HPA gallbladder glandular-cell pattern as a reference (HPA: High in gallbladder glandular cells). The selected-SKU caption identifies paraffin embedding but gives no fixative, so retrieval performance cannot establish formalin-fixed tissue performance (selected-SKU caption).
Could fixation be masking ADCY9 in my paraffin sections?
Target-specific fixation sensitivity is unknown because the selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption). Record the fixative, fixation time, section thickness, and retrieval conditions for each specimen before comparing staining intensity (standard IHC practice). If processing differs across specimens, run adjacent sections through the same citrate pH 6.0, 95–98 °C, 20 min retrieval and staining batch (page retrieval setting; standard IHC practice). Include a primary-antibody omission control, as in the catalog example, to identify detection-system signal (selected-SKU caption; standard IHC practice). Do not attribute a weak result to fixation alone when antibody concentration, section handling, and tissue preservation also vary (standard IHC practice).
Should ADCY9 stain membranes or cytoplasm in chromogenic IHC?
Expect possible membrane-associated signal because ADCY9 is annotated at the cell membrane and has 12 transmembrane segments (UniProt O60503 topology). Cytoplasmic staining is also reported in the selected-SKU paraffin image and across most tissues in HPA IHC (selected-SKU caption; HPA tissue IHC profile). Evaluate membrane rims and cytoplasmic signal separately, then compare each with staining in a primary-antibody omission control (standard IHC practice; selected-SKU caption). Strong nuclear-only staining would conflict with the reported localisation and warrants checking counterstain, background, and antibody-dependent signal (UniProt O60503 subcellular location; HPA subcellular; standard IHC practice). Interpret fine membrane detail cautiously where chromogenic deposits obscure cell boundaries (standard IHC practice).
How can epitope position change the ADCY9 staining I see?
ADCY9 has 12 annotated transmembrane segments, with long cytoplasmic regions at residues 302–786 and 997–1353 (UniProt O60503 topology). Its listed glycosylation sites at 206, 955, and 964 lie within annotated extracellular regions (UniProt O60503 topology and glycosylation). Check the catalog antibody’s stated immunogen or mapped epitope before interpreting a change in retrieval response or staining distribution (standard IHC practice). No isoforms are listed in the supplied record, so an unexpected pattern cannot be assigned to an annotated isoform from this evidence (UniProt O60503 isoforms). Compare adjacent sections under identical retrieval and detection conditions when assessing epitope accessibility (standard IHC practice).
How should I check ADCY9 localisation with multiplex immunofluorescence?
Pair ADCY9 with a marker for the cell population being examined, such as a glandular epithelial marker when assessing HPA-reported glandular staining (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with less tissue autofluorescence, then image unstained tissue to assess that background (standard IF practice). Because the catalog antibody’s epitope side is unspecified, test permeabilisation only after checking its immunogen: cytoplasmic epitopes generally require membrane access, whereas extracellular epitopes may be assessed without it (UniProt O60503 topology; standard IF practice). Compare matched permeabilised and unpermeabilised sections with single-label and primary-omission controls (standard IF practice). HPA reports supported plasma-membrane localisation and uncertain cytosolic localisation in its subcellular data (HPA subcellular).
What should I check when ADCY9 chromogenic staining is diffuse?
First compare the stained section with a primary-antibody omission control; the selected-SKU example used PBS in place of primary antibody (selected-SKU caption). If both show colour, check endogenous peroxidase blocking, secondary-reagent binding, and chromogen development time before changing retrieval (standard IHC practice). If only the primary-stained section is diffuse, titrate around the catalog example’s 1:50 dilution and inspect tissue edges and damaged areas separately (selected-SKU caption; standard IHC practice). Use identical counterstain and imaging settings when comparing these sections so differences in colour remain interpretable (standard IHC practice). Retain membrane and cytoplasmic patterns as distinct observations because both are represented in the supplied localisation evidence (UniProt O60503; HPA tissue IHC; selected-SKU caption).
How should I score ADCY9 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since HPA reports staining in specified cell types and the selected-SKU image reports cytoplasmic and membrane signal (HPA tissue IHC; selected-SKU caption). For a selected population, record percent positive cells and an H-score from 0–300 using intensity grades 0–3 (standard IHC practice). Score membrane and cytoplasm separately, and set thresholds with a primary-antibody omission control and the same imaging conditions (standard IHC practice). Normalise counts to evaluable cells, or stained-cell density to evaluable tissue area in mm², while excluding folds and necrotic regions (standard IHC practice). Report the antibody dilution, retrieval conditions, and scoring rules alongside results (standard IHC practice).
How can I distinguish true ADCY9 signal from tissue artefact?
A plausible signal follows the assessed cell population and membrane or cytoplasmic compartment: UniProt places ADCY9 at the cell membrane, while HPA tissue IHC reports widespread cytoplasmic expression (UniProt O60503; HPA tissue IHC). Compare candidate-positive cells with HPA’s High gallbladder glandular-cell and Not detected adipocyte observations, without treating either pattern as a universal control (HPA tissue IHC). Nuclear-only staining, staining restricted to cut edges or necrotic areas, and colour persisting without primary antibody warrant investigation as artefacts (reported localisation; standard IHC practice). Check endogenous peroxidase activity and secondary-reagent background before calling weak diffuse chromogen positive (standard IHC practice). HPA labels its tissue-IHC reliability Approved while noting medium staining–RNA consistency and pending external verification (HPA tissue IHC).
Boster reagents
Best ADCY9 / Adenylate cyclase type 9 IHC Antibodies
A06038-1 has real IHC images from paraffin sections of human breast and stomach carcinoma (catalog image captions). Human, mouse and rat reactivity is listed (catalog reactivity); IF data are absent (catalog IF image alts).
Real IHC data
Anti-Adenylate cyclase type 9 ADCY9 Antibody
Cat # A06038-1
A06038-1 is listed for IHC in human, mouse and rat (catalog applications/reactivity). Its IHC images show cytoplasmic and membrane staining in paraffin sections of human breast and stomach carcinoma at 1:50 (catalog image captions).
Which to pick: For tissue IHC, choose A06038-1, a rabbit polyclonal antibody with paraffin-section images from human breast and stomach carcinoma (catalog host/image captions). For IF/ICC, no SKU has a listed IF application or IF image (catalog applications/IF image alts). For mouse or rat IHC, A06038-1 has listed reactivity, while its depicted tissue data are human only (catalog reactivity/image captions); the fixative is unreported (catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O60503 (ADCY9_HUMAN, Adenylate cyclase type 9).
- Human Protein Atlas. ADCY9 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ADCY9 subcellular location (ICC-IF): Mainly localized to the plasma membrane and cytosol. In addition localized to the primary cilium and basal body..
- Human Protein Atlas. ADCY9 antibody validation summary (2 antibodies).
- Elevated Adenylyl Cyclase 9 Expression Is a Potential Prognostic Biomarker for Patients with Colon Cancer. Medical science monitor : international medical journal of experimental and clinical research 2018 — PMC5759510.
- ADCY9 functions as a novel cancer suppressor gene in lung adenocarcinoma. Journal of thoracic disease 2023 — PMC10089850.
- PubMed PMID:9628827 — UniProt-cited evidence.
- PubMed PMID:12972952 — UniProt-cited evidence.
- PubMed PMID:15879435 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ADCY9 (Adenylate cyclase type 9). Expected localisation Mostly cytoplasmic in tissue (HPA tissue IHC); membrane protein (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A06038-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Gallbladder (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A06038-1); 2. Fixation: Image fixative and duration unreported (datasheet A06038-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ADCY9, 1:50-1:200 (datasheet A06038-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Smooth muscle may be unstained despite reported airway expression (HPA tissue IHC; UniProt); Isoform / epitope: No isoforms annotated; epitope side of membrane is unspecified (UniProt); Regulation: Regulation of abundance is unreported (UniProt). Evidence: UniProt O60503, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the plasma membrane and cytosol. In addition…, Datasheet A06038-1, PMC5759510, PMC10089850. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ADCY9/immunohistochemistry-ihc-guide.